The 5 ' region of cnf1 harbours a translational regulatory mechanism for CNF1 synthesis and encodes the cell-binding domain of the toxin

The Escherichia coli cytotoxic necrotizing factor 1 (CNF1) is organized int o three functional domains: the N-terminal part containing the cell-binding domain, a putative central membrane-spanning region, and a C-terminal cata lytic region. On the basis of competition assays between CNF1 and GST-recom binant proteins containing different N-terminal fragments, and point mutati ons, we restricted the binding region to the first 190 amino acids. Hydroph ilic amino acids 53-75 are strictly necessary to cell receptor recognition. Using different cnf1-lacZ translational fusions, we demonstrated that the mRNA corresponding to the first 48 codons of cnf1 is involved in the transl ational regulation of CNF1 synthesis. This regulation consists of both a po sitive and a negative control. The positive control is exerted by codons 6- 20, including a putative downstream box that enhances the translational exp ression of cnf1. The negative control depends on codons 45-48. In this regi on, an anti-Shine-Dalgarno sequence, highly homologous to the core of the i nternal complementary sequence already reported for growth rate-regulated m etabolic genes, has been detected. To some extent, the inner structural org anization of CNF1 would thus suggest the compiling of several functions in a single mRNA protein system.